Lpt-723 and immune checkpoint inhibitor combinations and methods of treatment

ABSTRACT

The present invention provides, inter alia, a composition containing a compound of formula (I): 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt thereof, optionally, in combination with at least one immune checkpoint inhibitor compound. Kits containing the composition, and methods of using the composition for ameliorating or treating the effects of a disease such as a cancer, in a subject, are also provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/195,723, filed Jun. 28, 2016, which claims benefit to U.S.Provisional Application No. 62/186,157, filed Jun. 29, 2015. The entirecontents of the above applications are incorporated by reference as ifrecited in full herein.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains references to amino acids and/or nucleic acidsequences that have been filed concurrently herewith as sequence listingtext file “0563241.txt”, file size of 5 KB, created on Nov. 10, 2017.The aforementioned sequence listing is hereby incorporated by referencein its entirety pursuant to 37 C.F.R. § 1.52(e)(5).

FIELD OF THE INVENTION

The present disclosure relates generally to fields of cancer and cancertherapy. More specifically, the present disclosure relates tocompositions and methods comprising combinations of PI3Kγ inhibitors andat least one immune checkpoint inhibitor for the treatment of cancer.

BACKGROUND OF THE INVENTION

There is a well-known link between cancer and inflammation. Chronicinflammatory diseases are known to increase risk for developing tumors,and tumors provoke multiple inflammatory responses in order to avoidimmune system detection and destruction (Pardoll, 2012; Grivennikov etal., 2010). Cancers are able to exploit regulatory immune systemmechanisms, promoting angiogenesis, immunosuppression, and metastasis(Du et al., 2008; Lin et al., 2006; Bronte et al., 2000; Bunt et al.,2006; Kim et al., 2009). These regulatory mechanisms include theactivation of myeloid-derived suppressor cells (MDSCs) and immunecheckpoint pathways (Korman et al., 2006; Nagaraj et al., 2013; Talmadgeand Gabrilovich, 2013).

The disruption of immune checkpoints including programmed cell death-1(PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) usingantibodies to these receptors has shown promising responses in a subsetof patients in recent clinical trials (Nodi et al., 2010; Topalian etal., 2012; Brahmer et al., 2012; Wolchok et al., 2013). However, themajority of patients and tumor types in these trials remain unresponsiveto this immunotherapy. A study by Kim et al. (2014) indicated thatelevated levels of MDSCs may interfere with checkpoint inhibitortreatment by directly inhibiting the function of CD8+ T cells.Previously it was reported that blocking the PI3-kinase (PI3K) isoformp110γ can inhibit tumor inflammation, growth and metastasis bysuppressing MDSCs in a model of spontaneous breast cancer (Schmid,2011).

One report provides evidence of synergy between a pan-PI3K inhibitor andimmune checkpoint blockers to PD1/CTLA4 (Kim et al., 2014). That reportdemonstrated that reduction in MDSCs via PI3K inhibition contributes tosynergy with checkpoint blockade in syngeneic mouse breast and colontumor models. Pan-PI3K inhibitors have been recently tested in clinicaltrials, but they broadly inhibit all PI3K isorforms and have been shownto have many deleterious side effects (Cleary and Shapiro, 2010).

In view of the foregoing, there is a need for the development of new andbetter compositions for reducing MDSCs during immune checkpointblockade. The present invention is directing to meeting these and otherneeds.

SUMMARY OF THE INVENTION

Not wishing to be bound by any particular theory, it is believed thatpan-PI3K molecules reduce MDSCs through disruption of PI3Kγ isoformsignaling. PI3Kγ inhibitors are able to block tumor growth by inhibitingtumor inflammation and angiogenesis without directly affecting tumorcells (Schmid et al., 2011). In the present invention, a selective PI3Kγinhibitor is combined with immune checkpoint inhibitors and produces asynergistic anti-tumor effect.

Thus, the present invention provides a method for treating orameliorating the effects of a disorder in a subject comprisingadministering to the subject an effective amount of a first agent, whichis a compound of formula (I):

or a pharmaceutically acceptable salt thereof and a second agent, whichis an immune checkpoint inhibitor.

The present invention also provides a method for treating orameliorating the effects of a cancer in a subject comprisingadministering to the subject an effective amount of a compound offormula (I):

or a pharmaceutically acceptable salt thereof.

The present invention also provides a method for modulating a stromalmicroenvironment of a cancer comprising contacting the stromalmicroenvironment of the cancer with a compound of formula (I):

or a pharmaceutically acceptable salt.

The present invention also provides a composition for treating orameliorating the effects of a disorder in a subject, the compositioncomprising a first agent, which is a compound of formula (I):

or a pharmaceutically acceptable salt thereof and a second agent, whichis an immune checkpoint inhibitor.

The present invention also provides a kit comprising a first agent,which is a compound of formula (I):

or a pharmaceutically acceptable salt thereof and a second agent, whichis an immune checkpoint inhibitor, together with instructions for theiruse.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows a graph of median tumor volume for vehicle and checkpointinhibitor control groups in a syngeneic mouse colon cancer model(Colon26) with an intact immune system. Data is displayed for 57 days oftreatment. Group 1 mice (n=10) were given vehicle orally twice per dayfrom day 3 to endpoint and hamster polyclonal IgG intraperitoneally(i.p.) (100 μg/animal day 8; 50 μg/animal days 11 and 14). Group 2(n=10) received anti-CTLA-4 i.p. (100 μg/animal day 8; 50 μg/animal days11 and 14). Group 3 (n=10) received anti-PD-1 i.p. (100 μg/animalbiweekly starting day 3). Group 4 (n=5) received anti-PD-1 i.p. (100μg/animal biweekly starting day 3) and anti-CTLA-4 i.p. (100 μg/animalday 8; 50 μg/animal days 11 and 14).

FIG. 2 shows a graph of mean tumor volume for up to 75 days of treatmentin Colon26 mice. Groups 1-4 are control groups repeated from FIG. 1 forcomparison. Group 11 mice were given LPT-723 (MR237) orally twice perday at 10 mg/kg from day 3 to endpoint. Group 12 mice were given LPT-723orally twice per day at 30 mg/kg from day 3 to endpoint. Group 13received LPT-723 orally twice per day at 10 mg/kg from day 3 to endpointand anti-CTLA-4 i.p. (100 μg/animal day 8; 50 μg/animal days 11 and 14).Group 14 received LPT-723 orally twice per day at 30 mg/kg from day 3 toendpoint and anti-CTLA-4 i.p. (100 μg/animal day 8; 50 μg/animal days 11and 14). Group 15 received LPT-723 orally twice per day at 10 mg/kg fromday 3 to endpoint and anti-PD-1 i.p. (100 μg/animal biweekly startingday 3). Group 16 received LPT-723 orally twice per day at 30 mg/kg fromday 3 to endpoint and anti-PD-1 i.p. (100 μg/animal biweekly startingday 3). N=10 for all groups.

FIG. 3 shows a Kaplan-Meier survival plot of Groups 1-3 and 11-16 for 75days of treatment. Groups 13-16 received LPT-723 and immune checkpointinhibitor antibodies and are highlighted by arrows on the graph. Thegraph shows the percentage of animals surviving and provides evidence ofsynergy between LPT-723 and the checkpoint inhibitor antibodies that isnot present in either treatment alone.

FIG. 4 is a graph showing tumor volume in individual animals from Group2 during the course of the study. These animals received anti-CTLA-4i.p. (100 μg/animal day 8; 50 μg/animal days 11 and 14).

FIG. 5 is a graph showing tumor volume in individual animals from Group3 during the course of the study. These animals received anti-PD-1 i.p.(100 μg/animal biweekly starting day 3).

FIG. 6 is a graph showing tumor volume in individual animals from Group4 during the course of the study. These animals received anti-CTLA-4i.p. (100 μg/animal day 8; 50 μg/animal days 11 and 14) and anti-PD-1i.p. (100 μg/animal biweekly starting day 3).

FIG. 7 is a graph showing tumor volume in individual animals from Group11 during the course of the study. These animals received LPT-723 orallytwice per day at 10 mg/kg from day 3 to endpoint.

FIG. 8 is a graph showing tumor volume in individual animals from Group12 during the course of the study. These animals received LPT-723 orallytwice per day at 30 mg/kg from day 3 to endpoint.

FIG. 9 is a graph showing tumor volume in individual animals from Group13 during the course of the study. These animals received LPT-723 orallytwice per day at 10 mg/kg from day 3 to endpoint and anti-CTLA-4 i.p.(100 μg/animal day 8; 50 μg/animal days 11 and 14).

FIG. 10 is a graph showing tumor volume in individual animals from Group14 during the course of the study. These animals received LPT-723 orallytwice per day at 30 mg/kg from day 3 to endpoint and anti-CTLA-4 i.p.(100 μg/animal day 8; 50 μg/animal days 11 and 14).

FIG. 11 is a graph showing tumor volume in individual animals from Group15 during the course of the study. These animals received LPT-723 orallytwice per day at 10 mg/kg from day 3 to endpoint and anti-PD-1 i.p. (100μg/animal biweekly starting day 3).

FIG. 12 is a graph showing tumor volume in individual animals from Group16 during the course of the study. These animals received LPT-723 orallytwice per day at 10 mg/kg from day 3 to endpoint and anti-PD-1 i.p. (100μg/animal biweekly starting day 3).

FIG. 13 shows the median tumor volume over 15 days in a therapeuticColon26 cancer model. The purple line demonstrates the synergisticeffect of the combination of LPT-723 (30 mg/kg twice daily startingday 1) and anti-PD-1 antibody. N=10 animals per group.

FIG. 14 is a graph of median tumor volume in a Lewis lung cancer (LLC)syngeneic mouse model. LPT-723 (30 mg/kg twice daily starting day 2)combined with anti-PD-1 significantly inhibited tumor growth incomparison with anti-PD-1 alone. N=10 animals per group.

FIG. 15 is a graph of median tumor volume in the LLC syngeneic mousemodel. LPT-723 (30 mg/kg twice daily starting day 2) combined withanti-CTLA-4 significantly inhibited tumor growth in comparison withanti-CTLA-4 alone. N=10 animals per group.

FIG. 16A-FIG. 16B show the effects of LPT-723 in a Pan02 pancreaticcancer syngeneic mouse model. FIG. 16A is a graph showing median tumorvolume over 24 days for LPT-723 monotherapy and combination therapy withanti-PD-1. Group 1 (vehicle) received phosphate buffered saline (PBS)twice weekly i.p. and 0.2% MC/1% SLS in deionized (DI) water twice dailyorally. Group 2 received anti-PD-1 at 10 mg/kg twice weekly i.p. Group 3received LPT-723 at 30 mg/kg twice daily by mouth. Group 4 receivedLPT-723 and anti-PD-1 at the aforementioned doses. There were nosignificant changes in body weights. N=10 animals per group. FIG. 16Bshows an in vitro cell viability assay showing that LPT-723 does notdirectly kill Pan02 cells (IC₅₀=26.57 μM).

FIG. 17A-FIG. 17B show LPT-723 monotherapy and combination therapy in anA20 lymphoma cancer syngeneic model. FIG. 17A shows median tumor volumefor Groups 1-4 at the same dosing schedule as mice in FIG. 16. Therewere no significant changes in body weights. N=10 animals per group.FIG. 17B shows an in vitro cell viability assay showing that LPT-723does not directly kill A20 cells (IC₅₀=84.1 μM).

FIG. 18A-FIG. 18B show LPT-723 monotherapy and combination therapy in anMBT-2 bladder cancer syngeneic model. FIG. 18A shows median tumor volumefor Groups 1-4 at the same dosing schedule as mice in FIG. 16A. Therewere no significant changes in body weights. N=10 animals per group.FIG. 18B shows an in vitro cell viability assay showing that LPT-723does not directly kill MBT-2 cells (IC₅₀=45.49 μM).

FIG. 19A-FIG. 19B show LPT-723 monotherapy in a HCT116 colon xenograftmodel tested with vehicle and LPT-723 (30 mg/kg) only. LPT-723monotherapy activity demonstrated 45% tumor growth inhibition (FIG. 19A)despite no activity of LPT-723 up to 30 μM in vitro on HCT116 cells inculture (FIG. 19B). Tumor growth inhibition results suggest that LPT-723modulates the tumor stromal microenvironment rather than acting througha direct tumor cell effect.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present invention is a method for treating orameliorating the effects of a disorder in a subject comprisingadministering to the subject an effective amount of a first agent, whichis a compound of formula (I):

or a pharmaceutically acceptable salt thereof and a second agent, whichis an immune checkpoint inhibitor.

As used herein, the terms “treat,” “treating,” “treatment” andgrammatical variations thereof mean subjecting an individual subject toa protocol, regimen, process or remedy, in which it is desired to obtaina physiologic response or outcome in that subject, e.g., a patient. Inparticular, the methods and compositions of the present invention may beused to slow the development of disease symptoms or delay the onset ofthe disease or condition, or halt the progression of diseasedevelopment. However, because every treated subject may not respond to aparticular treatment protocol, regimen, process or remedy, treating doesnot require that the desired physiologic response or outcome be achievedin each and every subject or subject population, e.g., patientpopulation. Accordingly, a given subject or subject population, e.g.,patient population may fail to respond or respond inadequately totreatment.

As used herein, the terms “ameliorate”, “ameliorating” and grammaticalvariations thereof mean to decrease the severity of the symptoms of adisease in a subject.

The terms “administering”, “administration” and variants thereof(particularly “administering” a compound) as used herein in reference tothe combinations of the present invention means introducing thecomponents into the body of a subject, such as a human, in need of suchtreatment.

In some aspects of this and other embodiments the first and secondagents are administered as a single unit dose. In other aspects, thefirst and second agents are co-administered. In yet other aspects thefirst agent is administered prior to the second agent. In yet otheraspects the second agent is administered prior to the first agent.

In some aspects of this and other embodiments, the administration of thefirst and second agents to the subject provides a synergistic effect inthe treatment of the disorder.

As used herein, a “subject” is a mammal, preferably, a human. Inaddition to humans, categories of mammals within the scope of thepresent invention include, for example, farm animals, domestic animals,laboratory animals, etc. Some examples of farm animals include cows,pigs, horses, goats, etc. Some examples of domestic animals includedogs, cats, etc. Some examples of laboratory animals include primates,rats, mice, rabbits, guinea pigs, etc.

In some aspects of this and other embodiments, the subject is a mammal.Preferably, the mammal is selected from the group consisting of humans,primates, farm animals, and domestic animals. More preferably, themammal is a human.

In the present invention, an “effective amount” or a “therapeuticallyeffective amount” of an agent, monoclonal antibody, or fragment thereofor a compound or composition disclosed herein is an amount of suchmaterial that is sufficient to effect beneficial or desired results asdescribed herein when administered to a subject. Effective dosage forms,modes of administration, and dosage amounts may be determinedempirically, and making such determinations is within the skill of theart. It is understood by those skilled in the art that the dosage amountwill vary with the route of administration, the rate of excretion, theduration of the treatment, the identity of any other drugs beingadministered, the age, size, and species of mammal, e.g., human patient,and like factors well known in the arts of medicine and veterinarymedicine. In general, a suitable dose of any active agent disclosedherein or a composition containing the same will be that amount of theactive agent or composition, which is the lowest dose effective toproduce the desired effect.

A suitable, non-limiting example of a dosage of a compound of thepresent invention, and a monoclonal antibody, or an antigen bindingfragment disclosed herein is from about 1 mg/kg to about 2400 mg/kg perday, such as from about 1 mg/kg to about 1200 mg/kg per day, includingfrom about 50 mg/kg to about 1200 mg/kg per day. Other representativedosages of such agents include about 5 mg/kg, 10 mg/kg, 15 mg/kg, 20mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg,175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1100 mg/kg, 1200mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800mg/kg, 1900 mg/kg, 2000 mg/kg, 2100 mg/kg, 2200 mg/kg, and 2300 mg/kgper day. The effective dose of the compounds, antibodies, and antibodyfragments disclosed herein may be administered as two, three, four,five, six or more sub-doses, administered separately at appropriateintervals throughout the day.

As used herein, a “pharmaceutically acceptable salt” means a salt of thecompounds of the present invention which are pharmaceuticallyacceptable, as defined herein, and which possess the desiredpharmacological activity. Such salts include acid addition salts formedwith inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like; or withorganic acids such as acetic acid, propionic acid, hexanoic acid,heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,o-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,p-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,p-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid and the like. Pharmaceuticallyacceptable salts also include base addition salts which may be formedwhen acidic protons present are capable of reacting with inorganic ororganic bases. Acceptable inorganic bases include sodium hydroxide,sodium carbonate, potassium hydroxide, aluminum hydroxide and calciumhydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike.

The term “immune checkpoint inhibitor”, as used herein, refers to asubstance that blocks the activity of molecules involved in attenuatingthe immune response. Examples of immune checkpoint inhibitors include,but are not limited to inhibitors of CTLA-4, PD-1, LAG-3, B7-H3, B7-H4,TIM3, A2AR, and IDO.

In some aspects of this and other embodiments the immune checkpointinhibitor is selected from a group consisting of an anti-PD-1 antibody,an anti PD-L1 antibody, an anti-CTLA-4 antibody, and combinationsthereof. Preferably, the immune checkpoint inhibitor is selected from agroup consisting of nivolumab (Bristol-Myers Squibb), pembrolizumab(Merck), pidilizumab (Curetech), AMP-224 (GlaxoSmithKline/Amplimmune),MPDL3280A (Roche), MDX-1105 (Medarex, Inc./Bristol Myer Squibb),MEDI-4736 (Medimmune/AstraZeneca), arelumab (Merck Serono), ipilimumab(YERVOY, (Bristol-Myers Squibb), tremelimumab (Pfizer), pidilizumab(CureTech, Ltd.), IMP321 (Immutep S.A.), MGA271 (Macrogenics),BMS-986016 (Bristol-Meyers Squibb), lirilumab (Bristol-Myers Squibb),urelumab (Bristol-Meyers Squibb), PF-05082566 (Pfizer), IPH2101 (InnatePharma/Bristol-Myers Squibb), MEDI-6469 (MedImmune/AZ), CP-870,893(Genentech), Mogamulizumab (Kyowa Hakko Kirin), Varlilumab (CelIDexTherapeutics), Avelumab (EMD Serono), Galiximab (Biogen Idec), AMP-514(Amplimmune/AZ), AUNP 12 (Aurigene and Pierre Fabre), Indoximod (NewLinkGenetics), NLG-919 (NewLink Genetics), INCB024360 (Incyte) andcombinations thereof.

As used herein, an “antibody” and “antigen-binding fragments thereof”encompass naturally occurring immunoglobulins (e.g., IgM, IgG, IgD, IgA,IgE, etc.) as well as non-naturally occurring immunoglobulins,including, for example, single chain antibodies, chimeric antibodies(e.g., humanized murine antibodies), heteroconjugate antibodies (e.g.,bispecific antibodies), Fab′, F(ab′)₂, Fab, Fv, and rlgG. See, e.g.,Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford,Ill.); Kuby et al., 1998. As used herein, an “antigen-binding fragment”is a portion of the full length antibody that retains the ability tospecifically recognize the antigen, as well as various combinations ofsuch portions.

Non-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, can be produced recombinantly, or can be obtained,for example, by screening combinatorial libraries consisting of variableheavy chains and variable light chains as described by Huse et al.,Science 246:1275-1281 (1989), which is incorporated herein by reference.These and other methods of making, for example, chimeric, humanized,CDR-grafted, single chain, and bifunctional antibodies, are well knownto those skilled in the art (Winter and Harris, Immunol. Today14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow andLane, supra, 1988; Hilyard et al., Protein Engineering: A practicalapproach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed.(Oxford University Press 1995); each of which is incorporated herein byreference).

Full length antibodies can be proteolytically digested down to severaldiscrete, functional antibody fragments, which retain the ability torecognize the antigen. For example, the enzyme papain can be used tocleave a full length immunoglobulin into two Fab fragments and an Fcfragment. Thus, the Fab fragment is typically composed of two variabledomains and two constant domains from the heavy and light chains. The Fvregion is usually recognized as a component of the Fab region andtypically comprises two variable domains, one from each of the heavy(V_(H), “heavy chain variable region”, as used herein) and light (V_(L)“light chain variable region”, as used herein) chains. The enzyme pepsincleaves below the hinge region, so a F(ab′)₂ fragment and a pFc′fragment is formed. F(ab′)₂ fragments are intact antibodies that havebeen digested, removing the constant (Fc) region. Two Fab′ fragments canthen result from further digestion of F(ab′)₂ fragments. Examples ofantigen-binding fragments include, but are not limited to, Fv, Fab,Fab′, Fab′-SH, F(ab′)₂, diabodies, tribodies, scFvs, and single-domainantibodies (dAbs).

Typically, a full length antibody has at least one heavy and at leastone light chain. Each heavy chain contains a variable domain (V_(H)) andtypically three or more constant domains (C_(H)1, C_(H)2, C_(H)3, etc.),while each light chain contains a variable domain (V_(L)) and a constantdomain C_(L). Light and heavy chain variable regions contain four“framework” regions interrupted by three hypervariable regions, alsocalled “complementarity-determining regions” or “CDRs”. The extent ofthe framework regions and CDRs have been defined. See, e.g., Kabat etal., U.S. Dept. of Health and Human Services, Sequences of Proteins ofImmunological Interest (1983) and Chothia et al., J. Mol. Biol.196:901-917 (1987). The sequences of the framework regions of differentlight or heavy chains are relatively conserved within a species. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDRs in three dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody, whereas a V_(L) CDR1 is the CDR1 from thevariable domain of the light chain of the antibody.

The term “monoclonal antibody”, as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic epitope. The modifier “monoclonal” indicatesthe character of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made, e.g., by the hybridoma method firstdescribed by Kohler et al., Nature 256: 495 (1975), and as modified bythe somatic hybridization method as set forth above; or may be made byother recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

Additional types of antibodies that may be part of the monoclonalantibodies of the present invention include, but are not limited to,chimeric, humanized, and human antibodies. For application in man, it isoften desirable to reduce immunogenicity of antibodies originallyderived from other species, like mouse. This can be done by constructionof chimeric antibodies, or by a process called “humanization”. In thiscontext, a “chimeric antibody” is understood to be an antibodycomprising a domain (e.g. a variable domain) derived from one species(e.g. mouse) fused to a domain (e.g. the constant domains) derived froma different species (e.g. human).

As used herein, the term “humanized antibody” refers to forms ofantibodies that contain sequences from non-human (e.g., murine)antibodies as well as human antibodies. Such antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe framework (FR) regions are those of a human immunoglobulin sequence.The humanized antibody optionally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann etal., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol2:593-596 (1992)). Humanization can be essentially performed, e.g.,following the method of Winter and co-workers (Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-3′27 (1988);Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting rodentCDRs or CDR sequences for the corresponding sequences of a humanantibody.

Furthermore, technologies have been developed for creating antibodiesbased on sequences derived from the human genome, for example by phagedisplay or using transgenic animals (see, e.g., WO 90/05144; D. Marks,H. R. Hoogenboom, T. P. Bonnert, J. McCafferty, A. D. Griffiths and G.Winter (1991) “By-passing immunisation. Human antibodies from V-genelibraries displayed on phage.” J. Mol. Biol., 222, 581-597; Knappik etal., J. Mol. Biol. 296: 57-86, 2000; S. Carmen and L. Jermutus,“Concepts in antibody phage display”. Briefings in Functional Genomicsand Proteomics 2002 1(2):189-203; Lonberg N, Huszar D. “Human antibodiesfrom transgenic mice”. Int Rev Immunol. 1995; 13(1):65-93; Bruggemann M,Taussig M J. “Production of human antibody repertoires in transgenicmice”. Curr Opin Biotechnol. 1997 August; 8(4):455-8.). Such antibodiesare “human antibodies” in the context of the present invention.

As used herein, a “recombinant” antibody is any antibody whoseproduction involves expression of a non-native DNA sequence encoding thedesired antibody structure in an organism. In the present invention,recombinant antibodies include tandem scFv (taFv or scFv₂), diabody,dAb₂/VHH₂, knob-into-holes derivatives, SEED-IgG, heteroFc-scFv,Fab-scFv, scFv-Jun/Fos, Fab′-Jun/Fos, tribody, DNL-F(ab)₃, scFv₃-CH1/CL,Fab-scFv₂, IgG-scFab, IgG-scFv, scFv-IgG, scFv₂-Fc, F(ab′)₂-scFv₂,scDB-Fc, scDb-CH3, Db-Fc, scFv₂-H/L, DVD-Ig, tandAb, scFv-dhlx-scFv,dAb₂-IgG, dAb-IgG, dAb-Fc-dAb, and combinations thereof.

Variable regions of antibodies are typically isolated as single-chain Fv(scFv) or Fab fragments. ScFv fragments are composed of V_(H) and V_(L)domains linked by a short 10-25 amino acid linker. Once isolated, scFvfragments can be genetically linked with a flexible peptide linker suchas, for example, one or more repeats of Ala-Ala-Ala,Gly-Gly-Gly-Gly-Ser, etc. The resultant peptide, a tandem scFv (taFv orscFv₂) can be arranged in various ways, with V_(H)-V_(L) or V_(L)—V_(H)ordering for each scFv of the taFv. (Kontermann, R. E. In: BispecificAntibodies. Kontermann R E (ed.), Springer Heidelberg Dordrecht LondonNew York, pp. 1-28 (2011)).

As used herein, the term “epitope” refers to the portion of the antigenwhich is recognized by the antibody or antigen binding fragment. Asingle antigen (such as an antigenic polypeptide) may have more than oneepitope. Epitopes may be defined as structural or functional. Functionalepitopes are generally a subset of the structural epitopes and havethose residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.Epitopes formed from contiguous amino acids are typically retained onexposure to denaturing solvents, whereas epitopes formed by tertiaryfolding are typically lost on treatment with denaturing solvents.

In some aspects of this and other embodiments, the compound of formula(I) is selected from the group consisting of a substantially pureR-enantiomer thereof, a substantially pure S-enantiomer thereof, and aracemic mixture of the R- and S-enantiomers.

Preferably, the compound of formula (I) is a substantially pureR-enantiomer:

or a pharmaceutically acceptable salt thereof.

It is understood that the disclosure of a compound herein encompassesall stereoisomers of that compound. As used herein, the term“stereoisomer” refers to a compound made up of the same atoms bonded bythe same bonds but having different three-dimensional structures whichare not interchangeable. The three-dimensional structures are calledconfigurations. Stereoisomers include enantiomers, optical isomers, anddiastereomers.

The terms “racemate” or “racemic mixture” refer to a mixture of equalparts of enantiomers. The term “chiral center” refers to a carbon atomto which four different groups are attached. The term “enantiomericenrichment” as used herein refers to the increase in the amount of oneenantiomer as compared to the other.

It is appreciated that compounds of the present invention having achiral center may exist in and be isolated in optically active andracemic forms. Some compounds may exhibit polymorphism. It is to beunderstood that the present invention encompasses any racemic,optically-active, diastereomeric, polymorphic, or stereoisomeric form,or mixtures thereof, of a compound of the invention, which possess theuseful properties described herein, it being well known in the art howto prepare optically active forms (for example, by resolution of theracemic form by recrystallization techniques, by synthesis fromoptically-active starting materials, by chiral synthesis, or bychromatographic separation using a chiral stationary phase).

Examples of methods to obtain optically active materials are known inthe art, and include at least the following:

-   -   i) physical separation of crystals—a technique whereby        macroscopic crystals of the individual enantiomers are manually        separated. This technique can be used if crystals of the        separate enantiomers exist, i.e., the material is a        conglomerate, and the crystals are visually distinct;    -   ii) simultaneous crystallization—a technique whereby the        individual enantiomers are separately crystallized from a        solution of the racemate, possible only if the latter is a        conglomerate in the solid state;    -   iii) enzymatic resolutions—a technique whereby partial or        complete separation of a racemate by virtue of differing rates        of reaction for the enantiomers with an enzyme;    -   iv) enzymatic asymmetric synthesis—a synthetic technique whereby        at least one step of the synthesis uses an enzymatic reaction to        obtain an enantiomerically pure or enriched synthetic precursor        of the desired enantiomer;    -   v) chemical asymmetric synthesis—a synthetic technique whereby        the desired enantiomer is synthesized from an achiral precursor        under conditions that produce asymmetry (i.e., chirality) in the        product, which may be achieved using chiral catalysts as        disclosed in more detail herein or chiral auxiliaries;    -   vi) diastereomer separations—a technique whereby a racemic        compound is reacted with an enantiomerically pure reagent (the        chiral auxiliary) that converts the individual enantiomers to        diastereomers. The resulting diastereomers are then separated by        chromatography or crystallization by virtue of their now more        distinct structural differences and the chiral auxiliary later        removed to obtain the desired enantiomer;    -   vii) first- and second-order asymmetric transformations—a        technique whereby diastereomers from the racemate equilibrate to        yield a preponderance in solution of the diastereomer from the        desired enantiomer or where preferential crystallization of the        diastereomer from the desired enantiomer perturbs the        equilibrium such that eventually in principle all the material        is converted to the crystalline diastereomer from the desired        enantiomer. The desired enantiomer is then released from the        diastereomer;    -   viii) kinetic resolutions—this technique refers to the        achievement of partial or complete resolution of a racemate (or        of a further resolution of a partially resolved compound) by        virtue of unequal reaction rates of the enantiomers with a        chiral, non-racemic reagent or catalyst under kinetic        conditions;    -   ix) enantiospecific synthesis from non-racemic precursors—a        synthetic technique whereby the desired enantiomer is obtained        from non-chiral starting materials and where the stereochemical        integrity is not or is only minimally compromised over the        course of the synthesis;    -   x) chiral liquid chromatography—a technique whereby the        enantiomers of a racemate are separated in a liquid mobile phase        by virtue of their differing interactions with a stationary        phase. The stationary phase can be made of chiral material or        the mobile phase can contain an additional chiral material to        provoke the differing interactions;    -   xi) chiral gas chromatography—a technique whereby the racemate        is volatilized and enantiomers are separated by virtue of their        differing interactions in the gaseous mobile phase with a column        containing a fixed non-racemic chiral adsorbent phase;    -   xii) extraction with chiral solvents—a technique whereby the        enantiomers are separated by virtue of preferential dissolution        of one enantiomer into a particular chiral solvent;    -   xiii) transport across chiral membranes—a technique whereby a        racemate is placed in contact with a thin membrane barrier. The        barrier typically separates two miscible fluids, one containing        the racemate, and a driving force such as concentration or        pressure differential causes preferential transport across the        membrane barrier. Separation occurs as a result of the        non-racemic chiral nature of the membrane which allows only one        enantiomer of the racemate to pass through.

The stereoisomers may also be separated by usual techniques known tothose skilled in the art including fractional crystallization of thebases or their salts or chromatographic techniques such as LC or flashchromatography. The (+) enantiomer can be separated from the (−)enantiomer using techniques and procedures well known in the art, suchas that described by J. Jacques, et al., antiomers, Racemates, andResolutions”, John Wiley and Sons, Inc., 1981. For example, chiralchromatography with a suitable organic solvent, such asethanol/acetonitrile and Chiralpak AD packing, 20 micron can also beutilized to effect separation of the enantiomers.

The compound of formula (I) is referred to interchangeably as “LPT-723”.It is understood that the present invention also includes otherenantiomeric forms and racemic mixtures of LPT-723. The R isomer is apreferred agent.

In some aspects of this and other embodiments, the disorder is cancer,Preferably, the cancer is selected from the group consisting of bladdercancer, breast cancer, cervical cancer, colon cancer, esophageal cancer,endometrial cancer, gastric cancer, glioblastoma, head and neck cancer,hepatocellular carcinoma, leukemia, lung cancer, lymphoma, melanoma,multiple myeloma, neuroblastoma, neuroendocrine cancer, ovarian cancer,pancreatic cancer, prostate cancer, rectal cancer, renal cell carcinoma,rhabdoid cancer, sarcomas, and urinary track cancer. More preferably,the cancer is selected from the group consisting of bladder cancer,colon cancer, lung cancer, lymphoma, and pancreatic cancer.

Another embodiment of the present invention is a method for treating orameliorating the effects of a cancer in a subject comprisingadministering to the subject an effective amount of a compound offormula (I):

or a pharmaceutically acceptable salt thereof.

In some aspects of this and other embodiments, the cancer is selectedfrom the group consisting of bladder cancer, breast cancer, cervicalcancer, colon cancer, esophageal cancer, endometrial cancer, gastriccancer, glioblastoma, head and neck cancer, hepatocellular carcinoma,leukemia, lung cancer, lymphoma, melanoma, multiple myeloma,neuroblastoma, neuroendocrine cancer, ovarian cancer, pancreatic cancer,prostate cancer, rectal cancer, renal cell carcinoma, rhabdoid cancer,sarcomas, and urinary track cancer. Preferably, the cancer is coloncancer.

Another embodiment of the present invention is a method for modulating astromal microenvironment of a cancer comprising contacting the stromalmicroenvironment of the cancer with a compound of formula (I):

or a pharmaceutically acceptable salt.

As used herein, “tumors” and “cancers” are used interchangeably. Tumorsmay be benign or malignant. As used herein, the “stromalmicroenvironment” includes those stromal cells that are in a tumorcell's microenvironment and support the growth of tumor cells.

In this embodiment, “contacting” means bringing, e.g., LPT-723, theimmune checkpoint inhibitor, and/or one or more additional therapeuticagents into close proximity to the stromal microenvironment. This may beaccomplished using conventional techniques of drug delivery to mammalsor in the in vitro situation by, e.g., providing the LPT-723, the immunecheckpoint inhibitor, and/or one or more additional therapeutic agentsto a culture media in which the cancer cells are located.

Another embodiment of the present invention is a composition fortreating or ameliorating the effects of a disorder in a subject, thecomposition comprising a first agent, which is a compound of formula(I):

or a pharmaceutically acceptable salt thereof and a second agent, whichis an immune checkpoint inhibitor.

In some aspects of this and other embodiments, the composition is apharmaceutical composition further comprising a pharmaceuticallyacceptable carrier or diluent. In the present invention, all of thecompounds may be combined with pharmaceutically acceptable carriers ordiluents.

The compositions and pharmaceutical compositions of the presentinvention may be administered in any desired and effective manner: fororal ingestion, or as an ointment or drop for local administration tothe eyes, or for parenteral or other administration in any appropriatemanner such as intraperitoneal, subcutaneous, topical, intradermal,inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular,intravenous, intraarterial, intrathecal, or intralymphatic. Further, thecompositions and pharmaceutical compositions of the present inventionmay be administered in conjunction with other treatments. Eachcomposition and pharmaceutical composition of the present invention maybe encapsulated or otherwise protected against gastric or othersecretions, if desired.

The compositions and pharmaceutical compositions of the invention maycomprise one or more active ingredients in admixture with one or morepharmaceutically-acceptable diluents or carriers and, optionally, one ormore other compounds, drugs, ingredients and/or materials. Regardless ofthe route of administration selected, the agents/compounds of thepresent invention are formulated into pharmaceutically-acceptable dosageforms by conventional methods known to those of skill in the art. See,e.g., Remington, The Science and Practice of Pharmacy (21st Edition,Lippincott Williams and Wilkins, Philadelphia, Pa.).

Pharmaceutically acceptable diluents or carriers are well known in theart (see, e.g., Remington, The Science and Practice of Pharmacy (21stEdition, Lippincott Williams and Wilkins, Philadelphia, Pa.) and TheNational Formulary (American Pharmaceutical Association, Washington,D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, andsorbitol), starches, cellulose preparations, calcium phosphates (e.g.,dicalcium phosphate, tricalcium phosphate and calcium hydrogenphosphate), sodium citrate, water, aqueous solutions (e.g., saline,sodium chloride injection, Ringer's injection, dextrose injection,dextrose and sodium chloride injection, lactated Ringer's injection),alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol),polyols (e.g., glycerol, propylene glycol, and polyethylene glycol),organic esters (e.g., ethyl oleate and tryglycerides), biodegradablepolymers (e.g., polylactide-polyglycolide, poly(orthoesters), andpoly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils(e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut),cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones,talc, silicylate, etc. Each pharmaceutically acceptable diluent orcarrier used in a pharmaceutical composition of the invention must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the subject. Diluents orcarriers suitable for a selected dosage form and intended route ofadministration are well known in the art, and acceptable diluents orcarriers for a chosen dosage form and method of administration can bedetermined using ordinary skill in the art.

The compositions and pharmaceutical compositions of the invention may,optionally, contain additional ingredients and/or materials commonlyused in pharmaceutical compositions. These ingredients and materials arewell known in the art and include (1) fillers or extenders, such asstarches, lactose, sucrose, glucose, mannitol, and silicic acid; (2)binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, sodium starch glycolate, cross-linked sodiumcarboxymethyl cellulose and sodium carbonate; (5) solution retardingagents, such as paraffin; (6) absorption accelerators, such asquaternary ammonium compounds; (7) wetting agents, such as cetyl alcoholand glycerol monostearate; (8) absorbents, such as kaolin and bentoniteclay; (9) lubricants, such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, and sodium lauryl sulfate; (10)suspending agents, such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth;(11) buffering agents; (12) excipients, such as lactose, milk sugars,polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins,cocoa butter, starches, tragacanth, cellulose derivatives, polyethyleneglycol, silicones, bentonites, silicic acid, talc, salicylate, zincoxide, aluminum hydroxide, calcium silicates, and polyamide powder; (13)inert diluents, such as water or other solvents; (14) preservatives;(15) surface-active agents; (16) dispersing agents; (17) control-releaseor absorption-delaying agents, such as hydroxypropylmethyl cellulose,other polymer matrices, biodegradable polymers, liposomes, microspheres,aluminum monostearate, gelatin, and waxes; (18) opacifying agents; (19)adjuvants; (20) wetting agents; (21) emulsifying and suspending agents;(22), solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan; (23) propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane; (24) antioxidants; (25) agents which render theformulation isotonic with the blood of the intended recipient, such assugars and sodium chloride; (26) thickening agents; (27) coatingmaterials, such as lecithin; and (28) sweetening, flavoring, coloring,perfuming and preservative agents. Each such ingredient or material mustbe “acceptable” in the sense of being compatible with the otheringredients of the formulation and not injurious to the subject.Ingredients and materials suitable for a selected dosage form andintended route of administration are well known in the art, andacceptable ingredients and materials for a chosen dosage form and methodof administration may be determined using ordinary skill in the art.

Compositions and pharmaceutical compositions of the present inventionsuitable for oral administration may be in the form of capsules,cachets, pills, tablets, powders, granules, a solution or a suspensionin an aqueous or non-aqueous liquid, an oil-in-water or water-in-oilliquid emulsion, an elixir or syrup, a pastille, a bolus, an electuaryor a paste. These formulations may be prepared by methods known in theart, e.g., by means of conventional pan-coating, mixing, granulation orlyophilization processes.

Solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like) may be prepared, e.g., bymixing the active ingredient(s) with one or morepharmaceutically-acceptable diluents or carriers and, optionally, one ormore fillers, extenders, binders, humectants, disintegrating agents,solution retarding agents, absorption accelerators, wetting agents,absorbents, lubricants, and/or coloring agents. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using a suitable excipient. A tablet may be made by compressionor molding, optionally with one or more accessory ingredients.Compressed tablets may be prepared using a suitable binder, lubricant,inert diluent, preservative, disintegrant, surface-active or dispersingagent. Molded tablets may be made by molding in a suitable machine. Thetablets, and other solid dosage forms, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient therein.They may be sterilized by, for example, filtration through abacteria-retaining filter. These compositions may also optionallycontain opacifying agents and may be of a composition such that theyrelease the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.Any active ingredient of the invention can also be in microencapsulatedform.

Liquid dosage forms for oral administration includepharmaceutically-acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. The liquid dosage forms may containsuitable inert diluents commonly used in the art. Besides inertdiluents, the oral compositions may also include adjuvants, such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents. Suspensions maycontain suspending agents.

Compositions and pharmaceutical compositions of the present inventionfor rectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more active ingredient(s) withone or more suitable nonirritating diluents or carriers which are solidat room temperature, but liquid at body temperature and, therefore, willmelt in the rectum or vaginal cavity and release the active compound.Compositions and pharmaceutical compositions of the present inventionwhich are suitable for vaginal administration also include pessaries,tampons, creams, gels, pastes, foams or spray formulations containingsuch pharmaceutically-acceptable carriers as are known in the art to beappropriate.

Dosage forms for topical or transdermal administration include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches,drops and inhalants. The active agent(s)/compound(s) may be mixed understerile conditions with a suitable pharmaceutically-acceptable diluentor carrier. The ointments, pastes, creams and gels may containexcipients. Powders and sprays may contain excipients and propellants.

Compositions and pharmaceutical compositions of the present inventionsuitable for parenteral administrations comprise one or moreagent(s)/compound(s) in combination with one or morepharmaceutically-acceptable sterile isotonic aqueous or non-aqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain suitable antioxidants,buffers, solutes which render the formulation isotonic with the blood ofthe intended recipient, or suspending or thickening agents. Properfluidity can be maintained, for example, by the use of coatingmaterials, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants. These compositions mayalso contain suitable adjuvants, such as wetting agents, emulsifyingagents and dispersing agents. It may also be desirable to includeisotonic agents. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption.

In some cases, in order to prolong the effect of a drug (e.g., acomposition or a pharmaceutical composition of the present invention),it is desirable to slow its absorption from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material having poor watersolubility.

The rate of absorption of the active any active agent/composition of theinvention then depends upon its rate of dissolution which, in turn, maydepend upon crystal size and crystalline form. Alternatively, delayedabsorption of a parenterally-administered any active agent/compositionof the invention may be accomplished by dissolving or suspending theactive agent/composition in an oil vehicle. Injectable depot forms maybe made by forming microencapsule matrices of the active ingredient inbiodegradable polymers. Depending on the ratio of the active ingredientto polymer, and the nature of the particular polymer employed, the rateof active ingredient release can be controlled. Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue. The injectablematerials can be sterilized for example, by filtration through abacterial-retaining filter.

Any formulation of the invention may be presented in unit-dose ormulti-dose sealed containers, for example, ampules and vials, and may bestored in a lyophilized condition requiring only the addition of thesterile liquid diluent or carrier, for example water for injection,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the type described above.

In some aspects of this and other embodiments, the first and secondagents in the composition are in separate unit dose forms. In otheraspects, the first and second agents are in a single unit dose form.

Another embodiment of the present invention is a kit comprising a firstagent, which is a compound of formula (I):

or a pharmaceutically acceptable salt thereof and a second agent, whichis an immune checkpoint inhibitor, together with instructions for theiruse.

The kits may also include suitable storage containers, e.g., ampules,vials, tubes, etc., for each agent of the present invention (which maye.g., may be in the form of pharmaceutical compositions) and otherreagents, e.g., buffers, balanced salt solutions, etc., for use inadministering the agents to subjects. The agents of the invention andother reagents may be present in the kits in any convenient form, suchas, e.g., in a solution or in a powder form. The kits may furtherinclude a packaging container, optionally having one or more partitionsfor housing the pharmaceutical composition and other optional reagents.

Additional Definitions

As used herein, terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers, those containing modified residues, and non-naturallyoccurring amino acid polymers.

The term “amino acid” means naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,gamma-carboxyglutamate, and O-phosphoserine. An “amino acid analog”means compounds that have the same basic chemical structure as anaturally occurring amino acid, e.g., a carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. An “amino acid mimetic” means achemical compound that has a structure that is different from thegeneral chemical structure of an amino acid, but that functionssimilarly to a naturally occurring amino acid.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” used herein meanat least two nucleotides covalently linked together. Many variants of anucleic acid may be used for the same purpose as a given nucleic acid.Thus, a nucleic acid also encompasses substantially identical nucleicacids and complements thereof.

Nucleic acids may be single stranded or double stranded, or may containportions of both double stranded and single stranded sequences. Thenucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid may contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids may be synthesized as a single strandedmolecule or expressed in a cell (in vitro or in vivo) using a syntheticgene. Nucleic acids may be obtained by chemical synthesis methods or byrecombinant methods.

The nucleic acid may also be a RNA such as a mRNA, tRNA, short hairpinRNA (shRNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA),transcriptional gene silencing RNA (ptgsRNA), Piwi-interacting RNA,pri-miRNA, pre-miRNA, micro-RNA (miRNA), or anti-miRNA, as described,e.g., in U.S. patent application Ser. Nos. 11/429,720, 11/384,049,11/418,870, and 11/429,720 and Published International Application Nos.WO 2005/116250 and WO 2006/126040.

The nucleic acid may also be an aptamer, an intramer, or a spiegelmer.The term “aptamer” refers to a nucleic acid or oligonucleotide moleculethat binds to a specific molecular target. Aptamers are derived from anin vitro evolutionary process (e.g., SELEX (Systematic Evolution ofLigands by EXponential Enrichment), disclosed in U.S. Pat. No.5,270,163), which selects for target-specific aptamer sequences fromlarge combinatorial libraries. Aptamer compositions may bedouble-stranded or single-stranded, and may includedeoxyribonucleotides, ribonucleotides, nucleotide derivatives, or othernucleotide-like molecules. The nucleotide components of an aptamer mayhave modified sugar groups (e.g., the 2′-OH group of a ribonucleotidemay be replaced by 2′-F or 2′-NH₂), which may improve a desiredproperty, e.g., resistance to nucleases or longer lifetime in blood.Aptamers may be conjugated to other molecules, e.g., a high molecularweight carrier to slow clearance of the aptamer from the circulatorysystem. Aptamers may be specifically cross-linked to their cognateligands, e.g., by photo-activation of a cross-linker (Brody, E. N. andL. Gold (2000) J. Biotechnol. 74:5-13).

The term “intramer” refers to an aptamer which is expressed in vivo. Forexample, a vaccinia virus-based RNA expression system has been used toexpress specific RNA aptamers at high levels in the cytoplasm ofleukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA96:3606-3610).

The term “spiegelmer” refers to an aptamer which includes L-DNA, L-RNA,or other left-handed nucleotide derivatives or nucleotide-likemolecules. Aptamers containing left-handed nucleotides are resistant todegradation by naturally occurring enzymes, which normally act onsubstrates containing right-handed nucleotides.

A nucleic acid will generally contain phosphodiester bonds, althoughnucleic acid analogs may be included that may have at least onedifferent linkage, e.g., phosphoramidate, phosphorothioate,phosphorodithioate, or O-methylphosphoroamidite linkages and peptidenucleic acid backbones and linkages. Other analog nucleic acids includethose with positive backbones; non-ionic backbones, and non-ribosebackbones, including those disclosed in U.S. Pat. Nos. 5,235,033 and5,034,506. Nucleic acids containing one or more non-naturally occurringor modified nucleotides are also included within the definition ofnucleic acid. The modified nucleotide analog may be located for exampleat the 5′-end and/or the 3′-end of the nucleic acid molecule.Representative examples of nucleotide analogs may be selected fromsugar- or backbone-modified ribonucleotides. It should be noted,however, that also nucleobase-modified ribonucleotides, i.e.ribonucleotides, containing a non-naturally occurring nucleobase insteadof a naturally occurring nucleobase such as uridines or cytidinesmodified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromouridine; adenosines and guanosines modified at the 8-position, e.g.8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; O- andN-alkylated nucleotides, e.g. N6-methyl adenosine are suitable. The2′-OH-group may be replaced by a group selected from H, OR, R, halo, SH,SR, NH₂, NHR, NR₂ or CN, wherein R is C₁-C₆ alkyl, alkenyl or alkynyland halo is F, Cl, Br or I. Modified nucleotides also includenucleotides conjugated with cholesterol through, e.g., a hydroxyprolinollinkage as disclosed in Krutzfeldt et al., Nature (Oct. 30, 2005),Soutschek et al., Nature 432:173-178 (2004), and U.S. Patent ApplicationPublication No. 20050107325. Modified nucleotides and nucleic acids mayalso include locked nucleic acids (LNA), as disclosed in U.S. PatentApplication Publication No. 20020115080. Additional modified nucleotidesand nucleic acids are disclosed in U.S. Patent Application PublicationNo. 20050182005. Modifications of the ribose-phosphate backbone may bedone for a variety of reasons, e.g., to increase the stability andhalf-life of such molecules in physiological environments, to enhancediffusion across cell membranes, or as probes on a biochip. Mixtures ofnaturally occurring nucleic acids and analogs may be made;alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “an,” and“the” include plural referents unless the context clearly dictatesotherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitlycontemplated.

The following examples are provided to further illustrate the methods ofthe present invention. These examples are illustrative only and are notintended to limit the scope of the invention in any way.

EXAMPLES

The following examples are provided to further illustrate certainaspects of the present invention. These examples are illustrative onlyand are not intended to limit the scope of the invention in any way.

Example 1 Prophylactic Colon26 Colon Cancer Model:

Charles River (CR) BALB/c mice were implanted with Colon26 murine coloncancer cells on Day 1, and treatment was initiated on Day 3 with control(vehicle), anti-CTLA-4 (clone 4F10), anti-PD-1 (clone RMP1-14),anti-CTLA-4/anti-PD-1 combination, LPT-723 (10 mpk, 30 mpk),anti-CTLA-4/LPT-723 (10 mpk, 30 mpk) combination, or anti-PD-1/LPT-723(10 mpk, 30 mpk) combination. Anti-CTLA-4 was dosed i.p. on Day 8 at adose of 100 μg/animal and on Days 11, and 14 at a dose of 50 μg/animal.Anti-PD-1 was dosed i.p. twice a week for two weeks starting on Day 3 ata dose of 100 μg/animal. LPT-723 was dosed per os (p.o.) twice a daystarting on Day 3. Table 1 shows the response summary for groups 1-4:

Table 2 below shows the response summary for groups 1-3 and 11-16:

Very significant synergy was demonstrated in the groups withcombinations of LPT-723 (two dose cohorts) and immunotherapy checkpointinhibitors (PD-1 and CTLA-4 antibodies) showing decreased median tumorvolume (FIGS. 1-2) and increased survival (FIG. 3). Body weights rangedfrom 90-115% of baseline at the end of the study. Median tumor volumefor individual mice in control groups 2-4 and LPT-723 monotherapy andcombination therapy groups 11-16 are shown in FIGS. 4-12.

Therapeutic Colon26 Colon Cancer Model:

CR BALB/c mice bearing Colon26 murine syngeneic colon cancer weretreated with control (vehicle), anti-CTLA-4 (clone 9H10), anti-PD-1(clone RMP1-14), anti-CTLA-4/anti-PD-1 combination, LPT-723 (10 mpk, 30mpk), anti-CTLA-4/LPT-723 (10 mpk, 30 mpk) combination,anti-PD-1/LPT-723 (10 mpk, 30 mpk) combination, andanti-CTLA-4/anti-PD-1/LPT-723 (10 mpk, 30 mpk) combination. Treatmentwas initiated when tumor volume reached approximately 80-120 mm³.Anti-CTLA-4 was dosed i.p. on Day 2 at 100 μg/animal and on Days 5 and 8at 50 μg/animal. Anti-PD-1 was dosed i.p. twice a week for two weeks at100 μg/animal. LPT-723 was dosed p.o. twice a day starting Day 1.Results are shown for anti-PD-1, LPT-723, and anti-PD-1/LPT-723combination therapy groups indicating a synergistic effect betweenLPT-723 and anti-PD-1 (FIG. 13).

Example 2 Lewis Lung Cancer (LLC) Model:

C57/BL6 mice were implanted with Lewis Lung murine lung cancer cells onDay 1, and the treatment was initiated on Day 2 with control (vehicle),anti-CTLA-4 (clone 9H10), anti-PD-1 (clone RMP1-14),anti-CTLA-4/anti-PD-1 combination, LPT-723 (10 mpk, 30 mpk),anti-CTLA-4/LPT-723 (10 mpk, 30 mpk) combination, anti-PD-1/LPT-723 (10mpk, 30 mpk) combination, and anti-CTLA-4/anti-PD-1/LPT-723 (10 mpk, 30mpk) combination. Anti-CTLA-4 was dosed i.p. on Day 5 at 100 μg/animaland on Days 8 and 11 at 50 μg/animal. Anti-PD-1 was dosed i.p. twice aweek for two weeks starting on Day 2 at 100 μg/animal. LPT-723 was dosedp.o. twice a day starting on Day 2. LPT-723 at 30 mpk BID combined withanti-PD-1 (FIG. 14) or anti-CTLA-4 (FIG. 15) significantly inhibitedtumor growth in comparison with anti-PD-1 or anti-CTLA-4 alone in theLLC syngeneic mouse model.

Example 3 Pan02 Pancreatic Cancer Model:

C57/BL6 mice bearing Pan02 murine syngeneic pancreatic cancer weretreated with control (vehicle), anti-PD-1 (clone RMP1-14), LPT-723 andanti-PD-1/LPT-723 combination. Treatment was initiated when tumor volumereached approximately 80-120 mm³. Anti-PD-1 was dosed i.p. twice a weekfor three weeks at 10 mpk. LPT-723 was dosed p.o. twice a day at 30 mpk.

LPT-723 monotherapy and combination therapy resulted in greater than 60%decreased median tumor volume in this model (FIG. 16A). No significantchanges in body weights were noted. An in vitro cell viability assay(CellTiter-Glo luminescent assay) indicated that LPT-723 does notdirectly kill Pan02 cells (FIG. 16B, IC₅₀ for LPT-723 was 26.57 μM onPan02 cells versus 1.09 μM for the chemotherapeutic agent Cisplatin).

Example 4 A20 Lymphoma Cancer Model:

BALB/c mice bearing A20 murine syngeneic B cell lymphoma were treatedwith control (vehicle), anti-PD-1 (clone RMP1-14), LPT-723 andanti-PD-1/LPT-723 combination. Treatment was initiated when tumor volumereached approximately 80-120 mm³. Anti-PD-1 was dosed i.p. twice a weekfor three weeks at 10 mpk. LPT-723 was dosed p.o. twice a day at 30 mpk.LPT-723 monotherapy resulted in a 43% decrease in median tumor volumeand the combination therapy resulted in a 79% decrease at Day 15 (FIG.17A), indicating a highly synergistic effect of the combination. Nosignificant changes in body weights were noted. An in vitro cellviability assay indicated that LPT-723 does not directly kill A20 cells(FIG. 17B, IC₅₀ for LPT-723 was 84.10 μM on A20 cells versus 1.07 μM forCisplatin).

Example 5 MBT-2 Bladder Cancer Model:

C3H mice bearing MBT-2 murine syngeneic bladder cancer were treated withcontrol (vehicle), anti-PD-1 (clone RMP1-14), LPT-723 andanti-PD-1/LPT-723 combination. Treatment was initiated when tumor volumereached approximately 80-120 mm³. Anti-PD-1 was dosed i.p. twice a weekfor three weeks at 10 mpk. LPT-723 was dosed p.o. twice a day at 30 mpk.All therapy groups showed at least 70% reduction in median tumor volumecompared to control on Day 13 in the MBT-2 model (FIG. 18A). Nosignificant changes in body weights were noted. An in vitro cellviability assay indicated that LPT-723 does not directly kill MBT-2cells (FIG. 18B, IC₅₀ for LPT-723 was 45.49 μM on MBT-2 cells versus6.867 μM for Cisplatin).

Example 6 HCT-116 Human Colorectal Cancer Xenograft Model:

BALB/c nude mice bearing HCT-116 human colon xenografts were treatedwith control (vehicle) and LPT-723. Treatment was initiated when tumorvolume reached approximately 80-120 mm³. LPT-723 was dosed p.o. twice aday at 30 mpk. LPT-723 monotherapy activity was demonstrated, resultingin a 45% inhibition in tumor growth (FIG. 19A), despite no activity ofLPT-723 at doses up to 30 uM in vitro on HCT116 cells in culture (FIG.19B). The tumor growth inhibition results suggest LPT-723 modulates thetumor stromal microenvironment rather than a direct tumor cell effect.

Example 7 Upstate Kinase Panel:

The inhibitory activity of LPT-723 was determined by following theresidual kinase activity of many kinases which were tested either usinga radiometric assay, or a spectrophotometric assay. In the radiometricassay, radioactivity incorporated into the protein, phospho-peptide orphospho-lipid product from ³³P-□-ATP was followed as a function of time.In the spectrophotometric assay, each mole of ADP produced by the kinasereaction was coupled to the generation of one mole of NAD from NADHusing pyruvate kinase and lactate dehydrogenase.

The KinaseProfiler™ (Eurofins Pharma Discovery) assays employed asimilar radiometric detection assay. Table 3 below lists the peptidesubstrate, its concentration, and the ATP concentration used in eachkinase assay. The test compounds were introduced as DMSO solutions intothe assay. After a specified reaction time, the reaction was quenchedand the ³³P-labeled phospho-peptide/protein produced was trapped on afilter and quantified on a radioactivity counter. No toxicity issueswere observed.

TABLE 3 ATP ATP K_(M) [peptide Kinase Assay (μM) (μM) substrate]Substrate ABL  45   48  50 μM EAIYAAPFAKKK ALK 200  229 250 μMKKKSPGEYVNIEFG AMPKα1  90   80 200 μM AMARAASAAALSARRR ASK1 200  371  0.33 mg/mL MBP AXL  90   80 250 μM KKSRGDYMTMQIG BLK 120  125  0.1 mg/mL polyE4Y BTK 200 >200 250 μM KVEKIGEGTYGVVYK CaMKIIβ  15   22 30 μM KKLNRTLSV CDK1/CycB  45   51   0.1 mg/mL Histone H1 CDK5/P35  15  30   0.1 mg/mL Histone H1 CHK1  90   84 200 μM KKKVSRSGLYRSPSMPENLNRPRCK1γ1  45   38 200 μM KRRRALS(p)VASLPGL cKIT 200  450   0.1 mg/mLpolyE4Y cRAF 120  120   0.66 mg/mL MBP cSRC  45   45   0.1 mg/mL polyE4YEGFR  10    3   0.1 mg/mL polyE4Y EphB4  10   11   0.1 mg/mL polyE4Y FES 45   50   0.1 mg/mL polyE4Y FGFR3  15   28   0.1 mg/mL polyE4YFlt1 (VEGFR1) 200 2118 250 μM KKKSPGEYVNIEFG FYN  70   64 250 μMKVEKIGEGTYGVVYK HIPK2  10   10 0.33  mg/mL MBP IGF-1R 200 >200 250 μMKKKSPGEYVNIEFG IKKα  10    6 250 μM KKKKERLLDDRHDSGLDSMKDEE IR 200  447250 μM KKSRGDYMTMQIG JNK1α1  45   41 3 μM ATF2 LCK  90   90 250 μMKVEKIGEGTYGVVYK LYN  70   79 0.1 mg/mL polyE4Y MAPK1  70   67 250 μMPRELVEPLTPSGEAPNQALLR MEK1  10 ND 1 μM inactive MAPK2 MKK6  10 ND 1 μMinactive SAPK2a MKK7β  10 ND 2 μM inactive JNK1a1 MLCK  70   70 250 μMKKLNRTLSFAEPG MSK1  90   76 30 μM GRPRTSSFAEGKK MST2 155  165 0.33 mg/mLMBP mTOR  70   70 2 mg/mL mTOR substrate NEK2 120  120 0.33 mg/mL MBPPAK2  90   89 30 μM KEAKEKRQEQIAKRRLSSLRASTSKSGGSQK PDGFRα 120  1060.1 mg/mL polyE4Y Pim-2  15   15 300 μM RSRHSSPYAGT PKCβII  70   580.1 mg/mL Histone H1 PKCs 155  170 50 μM ERMRPRKRQGSVRRRV PKC6  15   160.1 mg/mL Histone H1 PRAK  15   18 30 μM KKLRRTLSVA PRK2  15   10 30 μMAKRRRLSSLRA RET  70   58 250 μM KKKSPGEYVNIEFG RIPK2 (RICK) 120  1330.33 mg/mL MBP ROCK II  15   22 30 μM KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQKROS (KROS) 200  563 250 μM KKKSPGEYVNIEFG RSK3  10    7 30 μM KKKNRTLSVASAPK2β  45   58 0.33 mg/mL MBP SGK  90   93 30 μM GRPRTSSFAEGKK TAK1  45  45 2 mg/mL casein TIE2 200  439 0.1 mg/mL polyE4Y TrkA 120  129 250 μMKKKSPGEYVNIEFG ZAP70  15   15 0.1 mg/mL polyE4Y Substrate SEQ ID NO.EAIYAAPFAKKK SEQ ID NO.: 1 KKKSPGEYVNIEFG SEQ ID NO.: 2 AMARAASAAALARRRSEQ ID NO.: 3 KKSRGDYMTMQIG SEQ ID NO.: 4 KVEKIGEGTYGVVYK SEQ ID NO.: 5KKLNRTLSVA SEQ ID NO.: 6 KKKVSRGLYRSPSMPENLNRPR SEQ ID NO.: 7KRRRALS(p)VASLPGL SEQ ID NO.: 8 KKKKERLLDDRHDSGLDSMKDEE SEQ ID NO.: 9RRELVEPLTPSGEAPNQALLR SEQ ID NO.: 10 KKLNRTLSFAEPG SEQ ID NO.: 11GRPRTSSFAEGKK SEQ ID NO.: 12 KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQKSEQ ID NO.: 13 RSRHSSYPAGT SEQ ID NO.: 14 ERMRPRKRQGSVRRRVSEQ ID NO.: 15 AKRRRLSSLRA SEQ ID NO.: 16

Example 8 2-Strain Ames Test:

Test System:

The two tester strains used were the Salmonella typhimurium histidineauxotrophs TA98 and TA100 as described by Ames et al. (1975). Testerstrain TA98 is reverted from auxotrophy to prototrophy by frameshiftmutagens while tester strain TA100 is reverted by mutagens that causeboth frameshift and base pair substitution mutations.

Experimental Design:

The test system was exposed to LPT-723 via the plate incorporationmethodology originally described by McCann et al. (1975) and updated byMaron and Ames (1983). Briefly, LPT-723 was tested at eight dose levelsalong with appropriate vehicle control and positive controls. Overnightcultures of tester strains TA98 and TA100 were plated on selectiveminimal agar in the presence and absence of Aroclor-induced rat liverS9. All dose levels of LPT-723, vehicle controls and positive controlswere plated in duplicate.

Plating and Scoring Procedures:

LPT-723 dilutions were prepared immediately before use and delivered tothe test system at room temperature under yellow light. One-half (0.5)milliliter of S9 or Sham mix, 100 μL of tester strain and 50 μL ofvehicle or LPT-723 dilution were added to 2.0 mL of molten selective topagar at 45±2° C. After vortexing, the mixture was overlaid onto thesurface of 25 mL of minimal bottom agar. When plating the positivecontrols, the LPT-723 aliquot was replaced by a 50 μL aliquot ofappropriate positive control. After the overlay had solidified, theplates were inverted and incubated for approximately 48 to 72 hours at37±2° C. Plates were counted immediately following the incubation periodor were stored at 2-8° C. until colony counting could be conducted.

The condition of the bacterial background lawn was evaluated forevidence of LPT-723 toxicity using a dissecting microscope. Precipitatewas evaluated by visual examination without magnification. Revertantcolonies for each tester strain and activation condition were countedeither entirely by automated colony counter or entirely by hand unlessthe plate exhibited toxicity. Plates with sufficient LPT-723 precipitateto interfere with automated colony counting were counted manually.

Solubility and Dose Levels Tested:

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based oncompatibility with the target cells. The LPT-723 formed solutions indimethyl sulfoxide (DMSO) from 0.03 to 100 mg/mL. The maximum dosetested was 5000 μg per plate, which dose was achieved using aconcentration of 100 mg/mL and a 50 μL plating aliquot. Dose levelstested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. Notoxicity was observed.

Example 9 Micronucleus Assay:

Prior to necropsy on Day 8, rats in a main study group of a 7-day oraltoxicity and toxicokinetic (TK) study were bled from the jugular veinfor the evaluation of the flow analysis of micronucleus. No toxicity wasobserved. The procedure used for collection and fixation of blood issummarized below:

Preparation of the Fixative Tubes:

Fixative tubes were prepared one day prior to blood collection. Two 15mL polypropylene centrifuge tubes (VWR no. 21008-103) were required persample. 2 mL of ultracold fixative-methanol was added to eachappropriately labeled tube (Tubes B1 and B2), and caps were replaced.The rack of tubes was placed overnight at approximately −80° C. to allowfor sufficient chilling of the fixative.

Preparation of the Anticoagulant/Diluent Vials (Prior to BloodCollection):

One vial (2 mL cryovials; VWR no. 66008-728) was required for eachsample. 350 μL of anticoagulant/diluent (Prototype Pig-a Mutation AssayAnticoagulant Solution, lot #17716, provided by BioReliance, fromLitron) was aseptically aliquoted into each appropriately labeled vial(Tube A). The vials were refrigerated (2-8° C.) until needed.

Collection of Blood Samples:

Approximately 0.3 mL of blood was collected from each animal into K₂EDTAtubes. The tubes were mixed well by inversion and placed on wet ice fora maximum of 30 minutes before proceeding. A volume of approximately 100μL (60 to 120 μL was required) of blood was transferred from the K₂EDTAtube and placed into the tube containing the cold anticoagulant (TubeA). The tubes containing the anticoagulant and blood (Tube A) were mixedgently by inversion and were kept on wet ice for a maximum of 30 minutesbefore fixing.

Fixing the Samples:

It was extremely important that the tubes containing the fixative andfixed blood remain ultra-cold (approximately −80° C.) and did not comein contact with vapors from dry ice. CO₂ vapor causes carbonation andcellular aggregation. For this same reason, the fixative was not storedin a freezer containing dry ice, and fixation was not performed on dryice. To avoid this problem, tubes containing the fixative were takendirectly from the freezer. The fixative was kept in an ultracold(approximately −80° C.) freezer. The following steps were performed veryquickly (in less than one minute) and were performed near the freezer.Each sample was fixed in duplicate. Duplicate (backup) samples areimportant in the event of shipping complications or if flow cytometricanalysis problems arise.

Immediately prior to fixing, the vial containing the blood/anticoagulantmixture (Tube A) was inverted to ensure a homogeneous suspension. Usinga micropipettor, 180 μL of blood was transferred from Tube A into eachof the duplicate tubes (Tubes B1 and B2) containing the ultra-coldfixative-methanol. The tip of the pipettor was held approximately 1 cmabove the fixative. Making sure that the pipette tip did not touch theside of the tube or the surface of the fixative, the 180 μL of dilutedblood sample was forcefully dispensed directly into the fixative. Thetube of fixed blood was capped tightly and vortexed briefly (only 3 to 5seconds) and returned to the ultra-cold freezer (approximately −80° C.).The samples were stored at approximately −80° C. (in a freezer where nodry ice was stored) until shipment. Samples (in Tubes B1 and B2) storedin the fixative at −70° C. or below are stable for at least 1 year, aslong as temperature is maintained. Remaining blood in the 2 mL cryovials(Tube A) containing cold anticoagulant was discarded.

Transferring of Fixed Samples into LTSS:

Samples were transferred into LTSS after they had been in the fixativefor at least 3 days. Each cryovial was labeled appropriately. The buffersolution was packed on ice to achieve ice-cold, but not freezing,temperature (approximately 45 minutes). With a container of ice and a 25mL pipette ready for aliquoting the buffer solution used below, thefollowing steps were performed as quickly as possible (withinapproximately 20 seconds). A tube of fixed blood sample (Tubes B1 andB2) was removed from the ultra-cold freezer. The capped tube was quicklyplaced on ice and the freezer was closed. The tube was vortexed for 3 to5 seconds to resuspend the cells and the cap was loosened on the tube.12 mL of ice-cold buffer solution was immediately added to each tube.Care was taken not to touch the tube with the pipette tip to preventtransfer of sample from one tube to another. The caps were tightened,the tubes inverted once to mix the solutions, and the tubes wereimmediately placed on ice until all were processed. Once the buffersolution had been added to the fixed cells, it was important that thetubes remained on ice or at 2° C. to 8° C. The tubes were centrifuged atapproximately 300×g to 400×g for 5 minutes. When centrifugation wascompleted, the tubes were quickly removed and immediately replaced onice. The supernatant from each tube was aspirated, leaving less than 50μL of supernatant in which to resuspend cells. Tubes were recapped andimmediately returned to ice. Working with one sample at a time, thecells were quickly resuspended in the remaining supernatant byvortexing. The tube was placed back on ice and the remaining sampleswere resuspended. 1 mL of long term storage solution (LTSS) (provided byBioReliance) was added to each tube, and the content was transferredinto the appropriate cryovial and the caps were tightened. The sampleswere stored at approximately −80° C. pending shipment. Test resultsrevealed no significant toxicity.

Example 10 Monkey Cardiovascular (CV) Studies:

Vehicle and LPT-723 Preparation & Experimental Design: The vehicle, 2%TPGS/1.5% HPMCAS-HF/1.5% PVP-VA with 50 mM pH 5.0 sodium citrate (indeionized water), was pre-formulated and was dispensed for use on eachday of dosing. The vehicle was stored stirring at room temperature in anamber glass container until acquired for dosing.

The test article LPT-723 was formulated as a 25% drug loaded spray drieddispersion with 75% of HPMC-AS as the inert excipient. Thus, testarticle formulation concentrations were calculated as the free baseusing a correction factor of 4. No adjustment was made for purity.Appropriate amounts of the LPT-723 were mixed with the vehicle toachieve nominal concentrations of 5 and 8 mg/mL. Formulations of LPT-723were prepared on each day of dosing as needed and were stored stirringat room temperature in an amber glass container until acquired fordosing.

Vehicle and LPT-723 Administration:

The same three or four male monkeys were administered vehicle (0 mg/kg)and LPT-723 at dose levels of 25 and 40 mg/kg via oral gavage at a dosevolume of 5 mL/kg (Table 4 below). All doses were administered accordingto a cross-over design, with one or two animals/dose level being dosedon each occasion with a 7 or 14-day washout period betweenadministrations. Animal number 6002 was not dosed at 25 mg/kg due to theanimal's transmitter no longer functioning properly. The formulationswere stirred throughout dose administration. After each dose and priorto removal of the gavage tube, the tube was flushed with 10 mL of tapwater. All cardiovascular and body temperature postdose data were basedon the tap water flush. Individual doses were based on the most recentbody weights. There were no significant toxicological observations.

TABLE 4 Experimental Design Number of Dose Dose Level Male Group (mg/kg)Animals^(a) 1 0 4 2 25  3^(b) 3 40 4 Dosing Schedule Animal Dose level(mg/kg) Number 0 25 40 6001 Days 1 and 22 Day 29 Day 8 6002 Days 1 and22 NA Day 8 6003 Days 8 and 29 Day 22 Day 1 6004 Days 8 and 29 Day 22Day 1 ^(a)Each dose group was administered to the same 3 or 4 animalsaccording to a cross-over design with at least a 7-day washout betweeneach dose. ^(b)Animal number 6002 was not dosed at 25 mg/kg due to anonfunctional transmitter. NA—Not applicable

Rat 28-Day GLP Toxicity Study:

Vehicle and Test Article Preparation:

The vehicle, 2% TPGS/1.5% HPMCAS-HF/1.5% PVP-VA with 50 mM pH 5.0 sodiumcitrate in deionized water, was prepared for use weekly and was storedat room temperature when not in use. A correction factor of 4 was usedto adjust for purity when preparing the LPT-723 formulations.Appropriate amounts of the LPT-723 were mixed with the vehicle toachieve nominal concentrations of 0.3, 0.6, and 1.2 mg/mL. Formulationsof the LPT-723 were prepared daily, stored at room temperature,protected from light, and were dosed within 4 hours of preparation.

Animal Acquisition and Acclimation:

A total of 98 male and 98 female CD® [Crl:CD®(SD)] rats (approximately 6to 7 weeks of age at receipt) were received from Charles RiverLaboratories, Portage, Mich. During the 10-day acclimation period, theanimals were observed daily with respect to general health and any signsof disease. All animals were given a detailed clinical examination priorto selection for study. The animals were administered a sham dose of tapwater on two occasions in the same manner and dose volume intended foruse on study.

Randomization, Assignment to Study, and Maintenance:

Using a standard, by weight, measured value randomization procedure, 85male and 85 female animals (weighing 228 to 283 g and 170 to 218 g,respectively, at randomization) were assigned to the control andtreatment groups identified in the following table.

TABLE 5 Group Assignments Group Dose Level Number of Animals Number(mg/kg/day) Male Female Main Study 1 0 15^(a) 15^(a) 2 3 15^(a) 15^(a) 36 15^(a) 15^(a) 4 12 15^(a) 15^(a) Toxicokinetic 5 0  4^(b)  4^(b) 6 3 7^(b)  7^(b) 7 6  7^(b)  7^(b) 8 12  7^(b)  7^(b) ^(a)Five animals weremaintained on study for a 4-week recovery peiod. ^(b)One additionalanimal was included as a possible replacement animal.

Vehicle and LPT-723 Administration:

Vehicle and LPT-723 were administered once daily for 22 days for mainstudy animals at 12 mg/kg/day, for 23 days for TK animals at 12mg/kg/day, and for 28 days for all animals at 0, 3, and 6 mg/kg/day. Theanimals were dosed via oral gavage at a dose volume of 10 mL/kg. Thecontrol group received the vehicle in the same manner as the treatedgroups. The vehicle and test article were dosed from stirredformulations at approximately the same time each day (±2 hours) based onthe first animal dosed each day. Individual doses were based on the mostrecent body weights.

Results:

The no observed adverse effect level (NOAEL) was <3 mg/kg. AUC wasapproximately 35 and therapeutic index was <4. The compound was poorlytolerated in rats: several clinical chemistry parameters were effected,most notably there was a large increase in circulating neutrophils. Theincreased neutrophil count correlated with tissue accumulation and aninflammatory state in multiple organs, particularly the gastrointestinaltract.

Example 12 Monkey 7-Day Dose Range Finding Study:

Preparation of the Control/Vehicle Item:

The control/vehicle item (2% TPGS/1.5% HPMCAS-HF/1.5% PVP-VA with 50 mMsodium citrate buffer pH 5.0) was prepared 3 days prior to the start ofdosing and was stored at room temperature, away from direct light forthe duration of the study. The instructions below were for thepreparation of a final volume of 2.5 L. These instructions were scaledas needed.

500 mL of 10% Vitamin E-TPGS were added to an appropriate sizedcontainer and stirring was initiated. 750 mL of 5% HPMCAS-HF/5% PVP-VAwere added to the same container and stirred at medium speed for 30minutes to ensure proper mixing. 1 L of deionized water was added andstirring was continued for 30 minutes. 125 mL of 1M sodium citratebuffer and 125 mL of deionized water were added with stirring. The finalvehicle was stirred at medium speed for 1 hour. The vehicle was a hazysolution and was stored at room temperature, away from direct light forthe duration of the study.

Experimental Design:

The test and control/vehicle items were administered once daily for 7consecutive days by oral gavage as described in Table 6 below. The dosevolume administered to each animal was 5 mL/kg. One day after the end ofdosing, the Main animals were euthanized and subjected to a necropsyexamination on Day 8.

TABLE 6 Dose Dose Main Group Group Dose Level Concentration VolumeAnimals Numbers Designation (mg/kg/day) (mg/mL) (mL/kg) Male Female 1Control* 0 0 5 2 2 2 Low Dose 15 3 5 2 2 3 Mid Dose 25 5 5 2 2 4 HighDose 40 8 5 2 2 *The Control animals received the control/vehicle item(2% TPGS/1.5% HPMCAS-HF/1.5% PVP-VA with 50 mM sodium citrate buffer pH5.0) alone. **Dose level and concentration are expressed in terms ofactive test item; consequently a correction factor of 4 was used whenweighing and dispensing the test item powder. LPT-723, was used as aspray dried dispersion (SDD) containing 25% active and 75% HPMCAS-HFpolymer.

Results:

The highest dose of LPT-723 exceeded the maximum tolerated dose (MTD).There was a dose-related decrease in lymphocyte counts with concomitanteffects in bone marrow and lymphoid tissues. Assuming a NOAEL atapproximately 50 AUC, the expected therapeutic index would beapproximately 5 with lower doses.

Example 13 Mouse 7-Day Dose Range Finding Study:

Preparation of Test and Control/Vehicle Items: Vehicle is 2% TPGS/1.5%HPMCAS-HF/1.5% PVP-VA with 50 mM sodium citrate (pH 5.0) in deionizedwater. LPT-723 is a spray dried dispersion (SDD) containing 25% activeand 75% HPMCAS-HF polymer. Test and vehicle items were stored at roomtemperature, protected from light, and desiccated. Dose formulation wasprepared daily. Unless indicated otherwise, LPT-723 was mixed withvehicle to achieve the desired concentrations, using appropriate mixingequipment to achieve homogenous formulations.

The LPT-723 and vehicle were administered once per day for 7 consecutivedays. Doses were administered at approximately the same time (±2 hours)throughout the duration of the study. All main study animals weresubmitted for necropsy on Day 8 (the day following the final dose). SeeTable 7 below.

TABLE 7 Number Dosing Information of Dose Dose Necropsy AnimalsLevel^(B) Conc.^(B) Dose Volume (Day 8) Group Male Treatment (mg/kg/day)(mg/mL) (mL/kg/day) Male Main Study Animals 1 6 Vehicle^(A) 0 0 10 6 2 6VRT-1098813 15 1.5 6 3 6 VRT-1098813 35 3.5 6 4 6 VRT-1098813 70 7 6Toxicokinetic Animals 5 18 VRT-1098813 15 1.5 10 — 6 18 VRT-1098813 353.5 — 7 18 VRT-1098813 70 7 — Conc.—concentration ^(A)Control animalsreceived vehicle: 2% TPGS/1.5% HPMCAS-HF/1.5% PVP-VA with 50 mM sodiumcitrate (pH 5.0) in deionized water ^(B)Dose level and concentration areexpressed in terms of active test item. A correction factor of 4.0 wasused when weighing and dispensing the LPT-723 powder. The test item,LPT-723 was used as a spray dried dispersion (SDD) containing 25% activeand 75% HPMCAS-HF polymer.

Results:

LPT-723 was well tolerated, with a decrease in lymphocyte and monocytecounts. There were no clinical signs or histopatholoy findings. Thisstudy provided additional mitigation of the apparently “rat specific”findings reported in Example 11.

CITED DOCUMENTS

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All patents, patent applications, and publications cited above areincorporated herein by reference in their entirety as if recited in fullherein.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention and all suchmodifications are intended to be included within the scope of thefollowing claims.

What is claimed is:
 1. A composition comprising: (1) a first agent,which is a compound of formula (I):

or a pharmaceutically acceptable salt thereof; and (2) a second agent,which is an immune checkpoint inhibitor.
 2. The composition of claim 1,wherein the compound of formula (I) is selected from the groupconsisting of a substantially pure R-enantiomer thereof, a substantiallypure S-enantiomer thereof, and a racemic mixture of the R- andS-enantiomers.
 3. The composition of claim 2, wherein the compound offormula (I) is a substantially pure R-enantiomer:


4. The composition according to claim 1, wherein the immune checkpointinhibitor is selected from a group consisting of an anti-PD-1 antibody,an anti PD-L1 antibody, an anti-CTLA-4 antibody, and combinationsthereof.
 5. The composition according to claim 1, wherein the immunecheckpoint inhibitor is selected from a group consisting of nivolumab(Bristol-Myers Squibb), pembrolizumab (Merck), pidilizumab (Curetech),AMP-224 (GlaxoSmithKline/Amplimmune), MPDL3280A (Roche), MDX-1105(Medarex, Inc./Bristol Myer Squibb), MEDI-4736 (Medimmune/AstraZeneca),arelumab (Merck Serono), ipilimumab (YERVOY, (Bristol-Myers Squibb),tremelimumab (Pfizer), pidilizumab (CureTech, Ltd.), IMP321 (ImmutepS.A.), MGA271 (Macrogenics), BMS-986016 (Bristol-Meyers Squibb),lirilumab (Bristol-Myers Squibb), urelumab (Bristol-Meyers Squibb),PF-05082566 (Pfizer), IPH2101 (Innate Pharma/Bristol-Myers Squibb),MEDI-6469 (MedImmune/AZ), CP-870,893 (Genentech), Mogamulizumab (KyowaHakko Kirin), Varlilumab (CelIDex Therapeutics), Avelumab (EMD Serono),Galiximab (Biogen Idec), AMP-514 (Amplimmune/AZ), AUNP 12 (Aurigene andPierre Fabre), Indoximod (NewLink Genetics), NLG-919 (NewLink Genetics),INCB024360 (Incyte) and combinations thereof.
 6. The compositionaccording to claim 1, wherein the composition is a pharmaceuticalcomposition further comprising a pharmaceutically acceptable carrier ordiluent.
 7. The composition according to claim 1, wherein the first andsecond agents are in a single unit dose form.
 8. A method for treatingor ameliorating the effects of cancer in a subject comprisingadministering to the subject an effective amount of a first agent,wherein the first agent is a selective PI3Kγ inhibitor orpharmaceutically acceptable salt thereof, and a second agent, whereinthe second agent is an immune checkpoint inhibitor.
 9. The methodaccording to claim 8, wherein the immune checkpoint inhibitor isselected from a group consisting of an anti-PD-1 antibody, an anti PD-L1antibody, an anti-CTLA-4 antibody, and combinations thereof.
 10. Themethod according to claim 8, wherein the immune checkpoint inhibitor isselected from a group consisting of nivolumab (Bristol-Myers Squibb),pembrolizumab (Merck), pidilizumab (Curetech), AMP-224(GlaxoSmithKline/Amplimmune), MPDL3280A (Roche), MDX-1105 (Medarex,Inc./Bristol Myer Squibb), MEDI-4736 (Medimmune/AstraZeneca), arelumab(Merck Serono), ipilimumab (YERVOY, (Bristol-Myers Squibb), tremelimumab(Pfizer), pidilizumab (CureTech, Ltd.), IMP321 (Immutep S.A.), MGA271(Macrogenics), BMS-986016 (Bristol-Meyers Squibb), lirilumab(Bristol-Myers Squibb), urelumab (Bristol-Meyers Squibb), PF-05082566(Pfizer), IPH2101 (Innate Pharma/Bristol-Myers Squibb), MEDI-6469(Medlmmune/AZ), CP-870,893 (Genentech), Mogamulizumab (Kyowa HakkoKirin), Varlilumab (CelIDex Therapeutics), Avelumab (EMD Serono),Galiximab (Biogen Idec), AMP-514 (Amplimmune/AZ), AUNP 12 (Aurigene andPierre Fabre), Indoximod (NewLink Genetics), NLG-919 (NewLink Genetics),INCB024360 (Incyte) and combinations thereof.
 11. The method accordingto claim 8, wherein the first and second agents are administered as asingle unit dose.
 12. The method according to claim 8, wherein the firstand second agents are co-adminstered.
 13. The method according to claim8, wherein the first agent is administered prior to the second agent.14. The method according to claim 8, wherein the second agent isadministered prior to the first agent.
 15. The method according to claim8, wherein the administration of the first and second agents to thesubject provides a synergistic effect in the treatment of the cancer.16. The method according to claim 8, wherein the cancer is selected fromthe group consisting of bladder cancer, breast cancer, cervical cancer,colon cancer, esophageal cancer, endometrial cancer, gastric cancer,glioblastoma, head and neck cancer, hepatocellular carcinoma, leukemia,lung cancer, lymphoma, melanoma, multiple myeloma, neuroblastoma,neuroendocrine cancer, ovarian cancer, pancreatic cancer, prostatecancer, rectal cancer, renal cell carcinoma, rhabdoid cancer, sarcomas,and urinary track cancer.
 17. The method according to claim 8, whereinthe cancer is selected from the group consisting of bladder cancer,colon cancer, lung cancer, lymphoma, and pancreatic cancer.
 18. Themethod according to claim 8, wherein the subject is a mammal.
 19. Themethod according to claim 18, wherein the mammal is selected from thegroup consisting of humans, primates, farm animals, and domesticanimals.
 20. The method according to claim 19, wherein the mammal is ahuman.
 21. The method according to claim 8, wherein the selective PI3Kγinhibitor is effective to reduce PI3Kγ kinase activity on one or more ofthe following protein substrates: ABL, ALK, AMPKα1, ASK1, AXL, BLK, BTK,CaMKIIβ, CDK1/CycB, CDEK5/P35, CHK1, CK1γ1, cKIT, cRAP, cSRC, EGFR,EphB4, FES, FGFR3, Flt1 (VEGFR1), FYN, HIPK2, IGF-1R, IKKα, IR, JNK1α,1,LCK, LYN, MAPK1, MEK1, MKK6, MKK7β, MLCK, MSK1, MST2, mTOR, NEK2, PAK2,PDGFRα, Pim-2, PKCβII, PKC₁, PKCθ, PRAK, PRK2, RET, RIPK2 (RICK), ROCKII, ROS (KROS), RSK3, SAPK2β, SGK, TAK1, TIE2, TrkA, and ZAP70.